Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

BACKGROUND: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. RESULTS: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. CONCLUSION: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

A mass spectrometric method for quantifying C3 and C6 phosphorylation of starch.

The glucosyl residues comprising starch can be phosphorylated at either the C3 or the C6 position of the molecule because of the activities of two distinct dikinase enzymes. After hydrolysis of the starch, the C6 phosphorylation is easy to measure using a routine enzyme assay for glucose 6-phosphate, but the C3 phosphorylation is more difficult to assay. A mass spectrometric (MS) method has been developed that, in a single run, can distinguish and quantify the glucose 3-phosphate and glucose 6-phosphate produced by hydrolysis of starch and can also measure the glucose content to give an accurate estimate of the starting material. The MS method involves quantification by LC/MS with external standards, using normal-phase hydrophilic interaction liquid chromatography and selective reaction monitoring. The MS method has been used to determine degrees of starch phosphorylation in a diverse group of potato lines, revealing threefold differences in phosphorylation between high- and low-phosphate lines. The method was also used to show that cold storage of potato tubers for up to 24weeks had little substantive effect on the levels of starch phosphorylation. MS provided an effective and efficient means of determining both the C6 and the C3 phosphorylation of starch.